Raquel Martín-Ibáñez

Brain-Derived Neurotrophic Factor Regulates the Onset and Severity of Motor Dysfunction Associated with Enkephalinergic Neuronal Degeneration in Huntington's Disease

The mechanism that controls the selective vulnerability of striatal neurons in Huntingtons disease is unclear. Brain-derived neurotrophic factor (BDNF) protects striatal neurons and is regulated by Huntingtin through the interaction with the neuron-restrictive silencer factor. Here, we demonstrate that the downregulation of BDNF by mutant Huntingtin depends on the length and levels of expression of the CAG repeats in cell cultures. To analyze the functional effects of these changes in BDNF in Huntingtons disease, we disrupted the expression of bdnf in a transgenic mouse model by cross-mating bdnf+/ - mice with R6/1 mice. Thus, we compared transgenic mice for mutant Huntingtin with different levels of BDNF. Using this double mutant mouse line, we show that the deficit of endogenous BDNF modulates the pathology of Huntingtons disease. The decreased levels of this neurotrophin advance the onset of motor dysfunctions and produce more severe uncoordinated movements. This behavioral pathology correlates with the loss of striatal dopamine and cAMP-regulated phosphoprotein-32-positive projection neurons. In particular, the insufficient levels of BDNF cause specific degeneration of the enkephalinergic striatal projection neurons, which are the most affected cells in Huntingtons disease. This neuronal dysfunction can specifically be restored by administration of exogenous BDNF. Therefore, the decrease in BDNF levels plays a key role in the specific pathology observed in Huntingtons disease by inducing dysfunction of striatal enkephalinergic neurons that produce severe motor dysfunctions. Hence, administration of exogenous BDNF may delay or stop illness progression.

Mutant huntingtin impairs post-Golgi trafficking to lysosomes by delocalizing optineurin/Rab8 complex from the Golgi apparatus.

Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.

Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor

BACKGROUND Human embryonic stem cells (hESCs) have potential use in clinical therapy and regenerative medicine. One of the major challenges regarding the application of these cells is the development of an efficient cryopreservation protocol, since current methods, which include slow-freezing-rapid thawing and vitrification of colonies in suspension, present poor viability and high differentiation rates. Dissociated hESC suspensions do not survive cryopreservation because they are susceptible to apoptosis upon cell detachment and dissociation. A selective Rho-associated kinase (ROCK) inhibitor has been reported to increase the survival of dissociated hESCs and their cloning efficiency. METHODS AND RESULTS Here, we describe a novel method for dissociated hESCs cryopreservation in the presence of the ROCK inhibitor Y-27632. The addition of this inhibitor to the freezing and post-thawing medium significantly increased the survival rate and efficiency of colony formation. Moreover, the hESC colonies obtained after the cryopreservation in the presence of the ROCK inhibitor showed a very low rate of differentiation and a reduced time of recovery. After prolonged culture of frozen-thawed dissociated hESCs, the characteristic properties of pluripotent cells were observed, including normal karyotype, morphological features, marker expression (SSEA-4, TRA-1-60, TRA-1-81 and Oct-4) and the potential to differentiate into derivatives of all three germ layers after embryoid bodies formation. CONCLUSION This novel method for the cryopreservation of dissociated hESCs may reduce the time required to amplify frozen stocks, and facilitate not only the storage of large numbers of hESCs but also the widespread use of these cells in regenerative medicine.

Interplay of leukemia inhibitory factor and retinoic acid on neural differentiation of mouse embryonic stem cells.

Embryonic stem (ES) cells have great potential for cell replacement in neurodegenerative disorders. Implantation of these cells into the brain, however, requires their prior differentiation. We examined the interplay between leukemia inhibitory factor (LIF) and retinoic acid (RA) on neural differentiation of mouse ES (mES) cells. Mouse embryonic stem cells were allowed to form cell aggregates, the so‐called embryoid bodies (EBs), in the absence or presence of LIF. In the absence of LIF, mES cells downregulated the expression of the undifferentiated mES cell marker Oct‐3/4, and increased mRNA levels of two neural precursor markers, Sox‐1 and Nestin, as well as the neuronal marker β‐tubulin III. This neuronal differentiation was enhanced by treating EBs with RA. Moreover, RA irreversibly increased the number of postmitotic neurons in culture, as shown by the reduction of proliferating mES cells and the increase in β‐tubulin III‐positive cells 6 days after RA removal, which in turn affected mES cell viability. The addition of LIF during EBs formation, however, blocked completely this neuronal differentiation. Our findings also showed that pre‐differentiation of mES cells in vitro avoided the teratocarcinoma formation observed when proliferating mES cells were grafted into the brain. In addition, mES cells pre‐differentiated with RA in culture showed a reduction in proliferation and the presence of neural phenotypes after grafting. In conclusion, the present results indicate that RA enhances neuronal differentiation of mES cells in the absence of LIF, although it compromises cell viability and transplantation.

Ikaros-1 couples cell cycle arrest of late striatal precursors with neurogenesis of enkephalinergic neurons.

During central nervous system development, several transcription factors regulate the differentiation of progenitor cells to postmitotic neurons. Here we describe a novel role for Ikaros‐1 in the generation of late‐born striatal neurons. Our results show that Ikaros‐1 is expressed in the boundary of the striatal germinal zone (GZ)/mantle zone (MZ), where it induces cell cycle arrest of neural progenitors by up‐regulation of the cyclin‐dependent kinase inhibitor (CDKi) p21Cip1/Waf1. This effect is coupled with the neuronal differentiation of late precursors, which in turn is critical for the second wave of striatal neurogenesis that gives rise to matrix neurons. Consistently, Ikaros−/− mice had fewer striatal projecting neurons and, in particular, enkephalin (ENK)‐positive neurons. In addition, overexpression of Ikaros‐1 in primary striatal cultures increases the number of calbindin‐ and ENK‐positive neurons. Our results also show that Ikaros‐1 acts downstream of the Dlx family of transcription factors, insofar as its expression is lost in Dlx1/2 double knockout mice. However, we demonstrate that Ikaros‐1 and Ebf‐1 independently regulate the final determination of the two populations of striatal projection neurons of the matrix compartment, ENK‐ and substance P‐positive neurons. In conclusion, our findings identify Ikaros‐1 as a modulator of cell cycle exit of neural progenitors that gives rise to the neurogenesis of ENK‐positive striatal neurons. J. Comp. Neurol. 518:329–351, 2010.

Arsenic and fluoride induce neural progenitor cell apoptosis

The aim of the present study is to determine the effect of inorganic arsenic (As) and its metabolites on the viability of the neural progenitor cell (NPC) line C17.2, in order to evaluate cellular mechanisms involved in As developmental neurotoxicity. Moreover, we analyzed the effects of the coexposure to As and fluoride (F), a situation to which some populations are commonly exposed. Our results show that NPCs are not susceptible to pentavalent As species [arsenate, monomethylarsonic acid, and dimethylarsinic acid] and F alone. However, the trivalent metabolites of arsenate [arsenite, monomethylarsonous acid, and dimethylarsinous acid] are toxic at concentrations below 1 mg/l, and this susceptibility increases when there is coexposure with F (≥ 5 mg/l). Arsenite triggers apoptosis after 24 h of exposure, whereas monomethylarsonous acid produces necrosis at very short times (2 h). Arsenite leads to an increase in intracellular Ca levels and generation of reactive oxygen species, which may cause a decrease in mitochondrial transmembrane potential, release of cytochrome c, and consequent activation of caspases. A slight activation of calpain also takes place, which might favor activation of the mitochondrial pathway or might activate other pathways. The treatment with some antioxidants such as quercetin and α-tocopherol shows only a partial reduction of the cytotoxicity.

Protective neuronal induction of ATF5 in endoplasmic reticulum stress induced by status epilepticus

Activating transcription factor 5 (ATF5) is a basic-leucine-zipper transcription factor of the ATF/CREB family. The Atf5 gene generates two transcripts, Atf5α and Atf5β, of which Atf5α is known to be selectively translated upon endoplasmic reticulum stress response in non-neuronal cells. ATF5 is highly expressed in the developing brain where it modulates proliferation of neural progenitor cells. These cells show a high level of ATF5 that has to decrease to allow them to differentiate into mature neurons or glial cells. This has led to the extended notion that differentiated neural cells do not express ATF5 unless they undergo tumourigenic transformation. However, no systematic analysis of the distribution of ATF5 in adult brain or of its potential role in neuronal endoplasmic reticulum stress response has been reported. By immunostaining here we confirm highest ATF5 levels in neuroprogenitor cells of the embryonic and adult subventricular zone but also found ATF5 in a large variety of neurons in adult mouse brain. By combining Atf5 in situ hybridization and immunohistochemistry for the neuronal marker NeuN we further confirmed Atf5 messenger RNA in adult mouse neurons. Quantitative reverse transcriptase polymerase chain reaction demonstrated that Atf5α is the most abundant transcript in adult mouse encephalon and injection of the endoplasmic reticulum stress inducer tunicamycin into adult mouse brain increased neuronal ATF5 levels. Accordingly, ATF5 levels increased in hippocampal neurons of a mouse model of status epilepticus triggered by intra-amygdala injection of kainic acid, which leads to abnormal hippocampal neuronal activity and endoplasmic reticulum stress. Interestingly, ATF5 upregulation occurred mainly in hippocampal neuronal fields that do not undergo apoptosis in this status epilepticus model such as CA1 and dentate gyrus, thus suggesting a neuroprotective role. This was confirmed in a primary neuronal culture model in which ATF5 overexpression resulted in decreased endoplasmic reticulum stress-induced apoptosis and the opposite result was achieved by Atf5 RNA interference. Furthermore, in vivo administration of the eIF2α phosphatase inhibitor salubrinal resulted in increased ATF5 hippocampal levels and attenuated status epilepticus-induced neuronal death in the vulnerable CA3 subfield. In good agreement with the neuroprotective effect of increased ATF5, we found that apoptosis-resistant epileptogenic foci from patients with temporal lobe epilepsy also showed increased levels of ATF5. Thus, our results demonstrate that adult neurons express ATF5 and that they increase its levels upon endoplasmic reticulum stress as a pro-survival mechanism, thus opening a new field for neuroprotective strategies focused on ATF5 modulation.

Nolz1 promotes striatal neurogenesis through the regulation of retinoic acid signaling

BackgroundNolz1 is a zinc finger transcription factor whose expression is enriched in the lateral ganglionic eminence (LGE), although its function is still unknown.ResultsHere we analyze the role of Nolz1 during LGE development. We show that Nolz1 expression is high in proliferating neural progenitor cells (NPCs) of the LGE subventricular zone. In addition, low levels of Nolz1 are detected in the mantle zone, as well as in the adult striatum. Similarly, Nolz1 is highly expressed in proliferating LGE-derived NPC cultures, but its levels rapidly decrease upon cell differentiation, pointing to a role of Nolz1 in the control of NPC proliferation and/or differentiation. In agreement with this hypothesis, we find that Nolz1 over-expression promotes cell cycle exit of NPCs in neurosphere cultures and negatively regulates proliferation in telencephalic organotypic cultures. Within LGE primary cultures, Nolz1 over-expression promotes the acquisition of a neuronal phenotype, since it increases the number of β-III tubulin (Tuj1)- and microtubule-associated protein (MAP)2-positive neurons, and inhibits astrocyte generation and/or differentiation. Retinoic acid (RA) is one of the most important morphogens involved in striatal neurogenesis, and regulates Nolz1 expression in different systems. Here we show that Nolz1 also responds to this morphogen in E12.5 LGE-derived cell cultures. However, Nolz1 expression is not regulated by RA in E14.5 LGE-derived cell cultures, nor is it affected during LGE development in mouse models that present decreased RA levels. Interestingly, we find that Gsx2, which is necessary for normal RA signaling during LGE development, is also required for Nolz1 expression, which is lost in Gsx2 knockout mice. These findings suggest that Nolz1 might act downstream of Gsx2 to regulate RA-induced neurogenesis. Keeping with this hypothesis, we show that Nolz1 induces the selective expression of the RA receptor (RAR)β without altering RARα or RARγ. In addition, Nozl1 over-expression increases RA signaling since it stimulates the RA response element. This RA signaling is essential for Nolz1-induced neurogenesis, which is impaired in a RA-free environment or in the presence of a RAR inverse agonist. It has been proposed that Drosophila Gsx2 and Nolz1 homologues could cooperate with the transcriptional co-repressors Groucho-TLE to regulate cell proliferation. In agreement with this view, we show that Nolz1 could act in collaboration with TLE-4, as they are expressed at the same time in NPC cultures and during mouse development.ConclusionsNolz1 promotes RA signaling in the LGE, contributing to the striatal neurogenesis during development.

Vesicular glutamate transporter 3 (VGLUT3) identifies spatially segregated excitatory terminals in the rat substantia nigra

The excitability of dopaminergic (DA) neurons in the substantia nigra is controlled by the convergent activity of multiple glutamatergic afferents. Here, we show that vesicular glutamate transporter 3 (VGLUT3)‐immunoreactive (ir) terminals segregate to the perisomatic region of DA neurons in the substantia nigra pars compacta, and VGLUT3 decorates a synapse population distinct from those marked by vesicular glutamate transporters 1 and 2. VGLUT3‐ir nerve endings form asymmetric terminals on DA neurons. Retrograde tracing suggests the superior colliculus as an origin of excitatory VGLUT3‐ir afferents. Collectively, our data indicate that VGLUT3 identifies a novel excitatory terminal subset that contributes to the tuning of DA cell excitability in the substantia nigra.

Helios Transcription Factor Expression Depends on Gsx2 and Dlx1&2 Function in Developing Striatal Matrix Neurons

Development of the nervous system is finely regulated by consecutive expression of cell-specific transcription factors. Here we show that Helios, a member of the Ikaros transcription factor family, is expressed in ectodermal and neuroectodermal-derived tissues. During embryonic development, Helios is expressed by several brain structures including the lateral ganglionic eminence (LGE, the striatal anlage); the cingulated, insular and retrosplenial cortex; the hippocampus; and the accessory olfactory bulb. Moreover, Helios is also expressed by Purkinje neurons during postnatal cerebellar development. Within the LGE, Helios expression follows a dynamic spatio-temporal pattern starting at embryonic stages (E14.5), peaking at E18.5, and completely disappearing during postnatal development. Helios is expressed by a small population of nestin-positive neural progenitor cells located in the subventricular zone as well as by a larger population of immature neurons distributed throughout the mantle zone. In the later, Helios is preferentially expressed in the matrix compartment, where it colocalizes with Bcl11b and Foxp1, well-known markers of striatal projection neurons. In addition, we observed that Helios expression is not detected in Dlx1/2 and Gsx2 null mutants, while its expression is maintained in Ascl1 mutants. These findings allow us to introduce a new transcription factor in the cascade of events that take part of striatal development postulating the existence of at least 4 different neural progenitors in the LGE. An Ascl1-independent but Gsx2- & Dlx1/2-dependent precursor will express Helios defining a new lineage for a subset of matrix striatal neurons.